An increasing number of clinical trials, e.g., cancer trials, require patient samples, e.g., tissue biopsies, to measure individual drug response markers [1]. For example, surgically harvested tissues are often used to collect data at two ends of the cellular spectrum: (i) genomic analyses that reveal driver oncogenes and specific mutations [2] and (ii) protein analyses of selected biomarkers intended to monitor cellular responses [3, 4]. Ideally, clinical samples are collected serially to monitor change in expression levels of key proteins. This raises many challenges, notably risk of morbidity with repeat core biopsies, increased cost, and logistical limitations. Alternative sample collection methods include fine-needle aspirates (FNAs), “liquid biopsies” of circulating tumor cells, or analysis of scant cells present in other easily harvested fluids. However, these samples have much lower cell numbers than biopsies, thereby limiting the number of proteins that can be analyzed.
After tissues have been sampled, selecting ubiquitous biomarkers can be difficult because of heterogeneity and dynamic network changes. Typically, small-molecule drugs influence more than one target proteins, whereas numerous proteins modulate downstream specific drug actions, trigger alternative molecular pathways, and induce tumor cell death or resistance [5]. The current tools to profile these key proteins in scant clinical samples are limited; standard practice encompasses immunocytology, which often precludes broad protein analysis because of insufficient sample within FNAs or liquid biopsies [6]. Thus, the number of markers is often limited (<10) and requires time-consuming analyses of tissue sections. Proteomic analyses by mass spectrometry remain technically challenging for single cells and phosphoproteomic detection and are costly for routine clinical purposes [7]. In research settings, multiplexed flow cytometry and mass cytometry have been used to examine an expanded set of markers (10 to 45) using single-cell populations. However, multiplexed flow cytometry often encounters limits in the amount of markers it can measure because of spectral overlap. Mass cytometry vaporizes cells during sample preparation, resulting in sample loss [8]. Accordingly, both of these existing methods do not enable isolating a rare cell of interest or performing concurrent genetic analyses once samples are used for proteomic analyses.
Hence, there remains a need for compositions and methods for simultaneous detection of a large number of target molecules from a sample.